Electroporation is a standard laboratory technique for transfection of bacteria, eukaryotic cells, and tissues in vivo. Our laboratories were among the first to develop in vivo plasmid electroporation for therapeutic applications by delivery to preclinical tumors, muscle, and skin and the first that conducted veterinary and clinical gene therapy trials. In vivo electroporation is becoming a well-accepted technique for clinical molecular delivery. Currently, nearly 100 clinical trials are registered in the NIH's clinicaltrials.gov database for electroporation of drugs and nucleic acids. We observed that complete tumor regression can occur when control backbone pDNA is electroporated into different tumors types using various electroporation protocols. Regression is preceded by the production of proinflammatory mRNAs and proteins. Our preliminary results implicate the activation of DNA-specific pattern recognition receptors (PRRs), which normally function for defense against pathogen invasion, in the induction of these proinflammatory proteins. These sensors are found in both immune and non-immune cell types. Activation of these sensors in non-immune cells, such as tumor cells, may contribute to the inflammation and regression that we observed in our previous research and may influence gene therapy efficacy. For example, inflammation may enhance the efficacy of an anti-tumor therapy or an infectious disease vaccine, but interfere with a protein replacement therapy targeting a healthy tissue. Our hypothesis is that DNA-specific PRR activation in non-immune cell types may influence the therapeutic potential of DNA electroporation. Therefore, in this proposal, we will elucidate the activation of PRRs in non-immune cells following different electroporation protocols in several normal cell types and in tumor cells, examine potential activation in a 3-dimensional microenvironment, and relate this pattern to subsequent in vivo effects. Our long-term goal is to be able to predict, based on putative PRR activation and in vivo effects, what electroporation parameters should be used for a specific tissue and therapeutic application. To test our hypothesis, we will conduct our experiments within the following specific aims: Specific Aim 1. Determine the DNA sensor activation pattern in response to plasmid DNA electroporation using various protocols in non-immune cell types Specific Aim 2. Determine effect of 3-dimensional culture and co-cultures of tumor and normal cells on the DNA sensor activation pattern in response to plasmid DNA electroporation Specific Aim 3. Determine the in vivo DNA sensor activation pattern after plasmid DNA electroporation and its potential influence on gene therapies delivered to tumor, muscle, and skin tissues.